1. Field of the Invention
This invention relates to the use of protein scaffolds for producing affinity reagents that specifically bind to phosphopeptides. The invention specifically relates to protein scaffolds produced from forkhead-associated domains that specifically bind to phosphopeptides comprising phosphorylated threonine amino acid residues. The invention in particular provides a plurality of phosphorylated peptide binding domains comprising libraries such as phage display libraries, methods for generating and isolating said phosphothreonine specific binding polypeptides, and methods for using said affinity reagents to monitor protein phosphorylation and study signaling events in cells.
2. Description of the Related Art
A cascade of signaling events, which involves many protein-protein interactions, is initiated within cells in response to external stimuli, including, for example, binding of a ligand to its receptor. In such cell signaling events, the signal is in many instances translocated to downstream effectors by the reversible action of protein kinases, phosphatases and phosphopeptide-binding domains. The human genome encodes for about 500 protein kinases and a third of that number of protein phosphatases (Manning et al. (2002) Science 298, 1912-34). Defective expression of kinases or phosphatases is the cause for various types of diseases (Cohen (2001) Eur J Biochem 268, 5001-10). Radioisotopic labeling studies have shown that a third of the total proteins in the cell are phosphorylated at any given time (Sefton et al. (2001) Curr Protoc Protein Sci Ch. 13, Unit 13 1. Phosphorylation of serine/threonine residues on proteins can lead not only to conformational changes in proteins but also create binding sites for phosphopeptide-binding domains, which play a critical role in the formation of multiprotein signaling complexes for relaying the signal to downstream signaling proteins (Yaffe et al. (2001) Curr Opin Cell Biol 113, 131-8). Similar to the recognition of phosphotyrosine (pY) residues by Src homology-2 (SH2) domains and phosphotyrosine binding (PTB) domains (Schlessinger et al. (2003) Sci STKE 2003, RE12; Yaffe (2002) Nat Rev Mol Cell Biol 3, 177-86), phosphorylation of serine/threonine residues creates binding sites for proteins containing phosphoserine/phosphothreonine (pS/pT)-binding domains, such as the 14-3-3 proteins (Muslin et al. (1996) Cell 84, 889-97; Bridges et al. (2005) Sci STKE 2005, RE10), tryptophan-tryptophan (WW) domain of Pin1 protein (Lu et al. (1999) Science 283, 1325-8), FHA domain found in prokaryotic and eukaryotic signaling proteins (Li et al. (2000) J Cell Sci 113 Pt 23, 4143-9; Tsai (2002) Structure 10, 887-8; Durocher et al. (2002) FEBS Lett 513, 58-66), and WD40 repeats of F-box proteins (Skowyra et al. (1997) Cell 91-209-19). These phosphoprotein-binding domains play a critical role in the formation of signaling complexes that eventually relay the extracellular signal downstream in the pathway. Therefore, it is evident that protein phosphorylation is a very important posttranslational modification, which is responsible for regulating proteins, translocating them to their proper subcellular location, and facilitating the formation of multiprotein complexes via protein interaction domains for transducing signals to downstream effectors and regulating processes such as gene expression, cytoskeletal rearrangements, cell cycle progression, DNA repair and apoptosis (Pawson et al. (2003)Science 300, 445-52; Mohammad et al. (2009) DNA Repair (Amst) 8, 1009-17).
Among the pS/pT-binding domains, the FHA domains are unique in that they recognize only pT containing peptides and do not show binding to either unphosphorylated threonine-containing or pS containing peptides (Durocher et al. (1999) Mol Cell 4, 387-94; Durocher et al. (2000) Mol Cell 6, 1169-82). The optimal binding motifs for various FHA domains, from Saccharomyces cerevisiae, Schizosaccharomyces pornbe, Arabidopsis thaliana and Mycobacterium tuberculosis were determined by using oriented phosphopeptide libraries that contain a fixed pT residue flanked by four degenerate residues on either side of it (Durocher et al. (2000) Mol Cell 6, 1169-82). From these screens, the pT +3 residue was found to be one of the major determinants of binding specificity. For example, the N-terminal FHA1 domain from S. cerevisiae Rad53 protein kinase prefers Asp at the pT +3 position (Liao et al. (2000) J. Mol. Biol 304, 941-51), the C-terminal FHA2 domain from the same protein prefers Leu/Iso at the pT+3 position (Byeon et al. (2001) J Mol Biol 314, 577-88) and Met/Leu/Phe at the pY +3 position (Wang et al. (2000) J Mol Biol 302, 927-40), and the FHA domain of the human Chk2 DNA damage check point kinase prefers Iso/Leu at the pT +3 position (Li et al. (2002) Mol Cell 9, 1045-54). The specificity of FHA domains ranges from recognizing singly or doubly phosphorylated sequences (Lee et al. (2008) Mol Cell 30, 767-78) to binding to an extended binding surface (Li et al. (2004) J Mol Biol 335, 371-81). From alanine-scanning experiments of the pT peptide, it was determined that the pT +3 residue contributed significantly to binding to the FHA1 domain (Durocher et al. (1999) Mol Cell 4, 387-94). Interestingly, non-conserved residues (G133 and G135) contribute to the pT +3 residue specificity (Yongkiettrakul et al. (2004) Biochemistry 43, 3862-9). The tightest FHA domain:pT peptide interaction (kd=100 nM) was recently reported with structural elucidation for specific pT vs. pS recognition (Pennell et al. (2010) Structure 18, 1587-95).
From previous structural studies on the FHA1-pT complex from various species, it was known that amino acid residues form four loops (i.e., β3-β4, β4-β5, β6-β7, and β10-β11) make contact with the pT peptide (Durocher et al. (2000) Mol Cell 6, 1169-82; Pennell et al. (2010) Structure 18, 1587-95; Mahajan et al. (2008) Sci Signal 1, re12). In FHA domains from different species, the β4-β5 loop varies in sequence, structure and length. For instance, the β4-β5 loop in ChK2 FHA domain contains 19 residues with a helical insertion in the loop. The length and structure of this loop determines its positioning either close to or away from the pT +3 residue, which is an important determinant of binding specificity: these sequence differences result in specific binding to phosphopeptides with either charged (for FHA1 domain) or hydrophobic (for ChK2 FHA domain) residues in the pT +3 position. Mutating residues in the 010-011 loop has been shown to alter the binding specificity of the FHA1 domain to be more like FHA2 (Yongkiettrakul et al. (2004) Biochemistry 43, 3862-9) and the amino acid residues in this loop may play an important role in the binding of the FHA1 domain to a pT peptide (from Mdt1 protein) containing a hydrophobic residue at the pT +3 position (Mahajan et al. (2005) J Am Chem Soc 127, 14572-3). Residues from the β6-β7 loop are known to be responsible for conferring preference for binding to pT- and not pS-containing peptides (Mahajen et al. (2008) Sci Signal 1, re12).
Antibodies recognizing phosphorylated residues in proteins can be valuable tools for studying phosphorylation of proteins upon cellular stimulation, for instance by epidermal growth factor (EGF) or insulin, and for unraveling biologically important signal transduction pathways. Antibodies recognizing phosphorylated residues in proteins are typically generated by immunizing animals with synthetic phosphopeptides (Sun et al. (2001) Biopolymers 60, 61-75; Bangalore et al. (1992) Proc Natl Acad Sci USA 89, 11637-41). However, thousands of phosphorylation sites exist in the human proteome, so that conventional methods for generating anti-phosphopeptide antibodies would require immunization with a specific phosphopeptide for each phosphorylation site in the human proteome, making this process time consuming, expensive, laborious and impractical.
An alternative method for generating antibodies to phosphoproteins is to use recombinant methods to generate specific binding peptides or polypeptides, including antibodies in less time. For example, antibody fragments and various engineered proteins (Gebauer et al. (2009) Curr Opin Chem Biol 13, 245-55) have been exploited as scaffolds for generating useful affinity reagents.